Figure 4.

Substrate specificity of recombinant AtGCD3. A and B, the recombinant AtGCD3 hydrolyzes C16 long-chain fatty acid GlcCer to C16 long-chain fatty acid hydroxyceramide in naturally occurring lipid substrates extracted from Arabidopsis leaves. Natural glucosylceramides were extracted from Arabidopsis, incubated with (B) or without (A) recombinant AtGCD3 at 30 °C for 24 h, and then subjected to HPLC ESI-MS/MS analysis. Each peak (a, b, c, d, e, and f) was further subjected to MS/MS, as shown in Fig. 5. C and D, quantitative analysis of hydrolysis using Arabidopsis leaf lipids. Naturally occurring glucosylceramides (C) were hydrolyzed to hydroxyceramides (D). The inset in D indicates that Glc-d18:0-h16:0 was hydrolyzed to d18:0-h16:0. E and F, the recombinant AtGCD3 preferentially hydrolyzes long-acyl-chain GlcCer. 500 nm commercially available GlcCers with different fatty acid chain length were incubated with (+) or without (−) AtGCD3 at 30 °C for 13 h, respectively, and analyzed by HPLC ESI-MS/MS. Commercial glucosylceramides (E) were hydrolyzed to ceramides (F). G, the recombinant AtGCD3 has no activity on galactosylceramide and MGDG. 500 nm Gal-d18:1–16:0 or MGDG was incubated with (+) or without (−) AtGCD3 at 30 °C for 13 h and analyzed by HPLC ESI-MS/MS. Data represent the mean ± S.D. (error bars) (n = 3) of independent experiments. The statistical significance of differences was determined with (+) or without (−) AtGCD3 in each GlcCer by Student's t test; p values are indicated where p < 0.05.